Solution for extraction of rna

ABSTRACT

A solution for extracting substantially pure RNA from a biological sample is disclosed. The solution for extracting RNA from a biological sample containing RNA and at least DNA comprises:
         (a) phenol in an amount of more than 50% by volume based on the total amount of the solution;   (b) a polyol in an amount of 3 to 10% by volume based on the total amount of the solution;   (c) a guanidinium salt at a concentration of 0.5 to 2.0 M based on the total amount of the solution;   (d) a thiocyanate at a concentration of 0.1 to 0.5 M based on the total amount of the solution; and   (e) a buffer for maintaining the pH of the solution at 4 to 6.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase application of PCT InternationalApplication No. PCT/JP2011/068620, filed Aug. 17, 2011, and claimspriority to Japanese Patent Application No. 2010-183280, filed Aug. 18,2010, the disclosures of both applications are incorporated herein byreference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to a solution for extracting substantiallypure RNA from a biological sample.

BACKGROUND OF THE INVENTION

Genetic information written in DNA is transcribed into RNA in variouscombinations, to produce complicated phenotypes of organisms.Contribution of RNA to phenotypes of organisms is known to be dependenton the types and expression levels of the RNA, and extraction of highlypure RNA from various biological materials is important for performinggene expression analysis. For achievement of this object, many methodsfor extraction of RNA have been developed so far. Examples of methodsfor isolation of RNA frequently employed include phenol extraction,precipitation from chaotropic salt solutions and adsorption to silicamembranes.

Patent Document 1 discloses a solution for RNA extraction comprising 2to 5 M guanidine and 40 to 60% phenol. RNA extraction had required notless than 2 days of operation using an ultracentrifuge before, but useof this solution enabled efficient extraction of RNA in 3 hours. Thismethod is called the single-step method.

By improvement of the method described in the above Patent Document 1,Patent Document 2 discloses an extraction solution for simultaneousextraction and separation of RNA, DNA and proteins from a samplecomprising of these components. More specifically, the literaturedescribes extraction and separation of RNA into an aqueous layer byusing a 30 to 50% phenol solution containing 0.5 to 2 M guanidine.

Although the solutions described in Patent Documents 1 and 2 havedifferent compositions, RNA can be extracted by similar operations usingthe solutions. That is, each solution is used for homogenization of abiological tissue, and a hydrophobic organic solvent such as chloroformis used upon centrifugation of the homogenate to achieve layerseparation. Thereafter, the aqueous layer in the uppermost partcomprising RNA is recovered. RNA is then precipitated with alcohol andwashed in order to extract RNA.

However, RNA isolated using the solutions and the methods described inPatent Document 1 and 2 still shows contamination with (residual)genomic DNA in an amount which can be detected by the reversetranscription-polymerase chain reaction assay (RT-PCR), leading toproblems such as loss of quantitativeness of RNA in cases of RT-PCR(Patent Document 3, e.g., paragraph 0005). Therefore, RNA isolated bythese methods needs to be further purified for removal of DNA as acontaminant.

A commonly used method for removal of DNA contained as an impurity in anextracted RNA sample is treatment of the RNA sample withdeoxyribonuclease (DNase). However, in cases where treatment with DNaseis carried out in a liquid layer, it is necessary to performphenol/chloroform extraction and denaturation of proteins again forremoval of DNase after the treatment. Further, in cases where theextraction is performed using a combination of silica membrane columns,the operation of washing the columns needs to be carried out repeatedly.Although contamination with DNA is reduced by this treatment with DNase,such additional labor is required and loss of RNA occurs, resulting in adecreased amount of extracted RNA, which is problematic.

As a method for avoiding contamination of an RNA sample with DNA withoutperforming DNase treatment, Patent Document 3 reports a method using anRNA extraction reagent at a pH of less than 4. However, it is well knownthat nucleic acid is depurinated and degraded under acidic conditions,and it is therefore difficult to isolate substantially intact RNA.Further, since the solution equilibrium of DNA into the aqueous/organiclayer under acidic conditions is biased toward distribution into theorganic layer, the effect of suppressing contamination of the aqueousphase with genomic DNA can be expected to some extent by using a reagentfor extraction of RNA at a pH of less than 4, but complete suppressionof contamination with small DNA fragments having small numbers of basesis impossible.

PATENT DOCUMENTS

-   Patent Document 1: U.S. Pat. No. 4,843,155 B-   Patent Document 2: JP 5-344886 A-   Patent Document 3: Japanese Translated PCT Patent Application    Laid-open No. 2007-532140

SUMMARY OF THE INVENTION

As described above, substantially pure RNA free from contamination withDNA cannot be extracted with conventional solutions for extraction ofRNA from biological samples even in cases where quantitativeness isrequired, which has been problematic. Therefore, for removal of DNA as acontaminant, an additional step such as DNase treatment has beennecessary. The present invention aims to solve these problems andprovides a solution for extracting substantially pure RNA from abiological sample.

The present inventors studied compositions of conventional solutions forRNA extraction and discovered that the phenol concentration has anespecially strong relationship with the effect of prevention ofcontamination with DNA, thereby completing the present invention.

That is, the present invention provides the following:

-   [1] A solution for extracting RNA from a biological sample    containing RNA and at least DNA, the solution comprising:

(a) phenol in an amount of more than 50% by volume based on the totalamount of the solution;

(b) a polyol in an amount of 3 to 10% by volume based on the totalamount of the solution;

(c) a guanidinium salt at a concentration of 0.5 to 2.0 M based on thetotal amount of the solution;

(d) a thiocyanate at a concentration of 0.1 to 0.5 M based on the totalamount of the solution; and

(e) a buffer for maintaining the pH of the solution at 4 to 6.

-   [2] The solution according to [1], wherein the phenol concentration    is 55 to 65% by volume based on the total amount of the solution.-   [3] The solution according to [1] or [2], further comprising an    organic solvent for separating an aqueous layer.-   [4] The solution according to any one of [1] to [3], wherein the    biological sample is a culture liquid of cultured cells.-   [5] The solution according to any one of [1] to [3], wherein the    biological sample is a body fluid component of an organism.-   [6] The solution according to any one of [1] to [3], wherein the    biological sample is a blood component of an organism-   [7] A method for extracting RNA from a biological sample containing    RNA and at least DNA, the method comprising the steps of:

homogenizing the biological sample together with a solution comprising:

-   -   (a) phenol in an amount of more than 50% by volume based on the        total amount of the solution;    -   (b) a polyol in an amount of 3 to 10% by volume based on the        total amount of the solution;    -   (c) a guanidinium salt at a concentration of 0.5 to 2.0 M based        on the total amount of the solution;    -   (d) a thiocyanate at a concentration of 0.1 to 0.5 M based on        the total amount of the solution; and    -   (e) a buffer for maintaining the pH of the solution at 4 to 6;

mixing the obtained homogenate with an organic solvent for separation ofan aqueous layer;

centrifuging the obtained mixture; and

recovering an RNA-containing aqueous layer produced by thecentrifugation.

-   [8] A method for extracting RNA from a biological sample containing    RNA and at least DNA, the method comprising the steps of:

homogenizing the biological sample together with a solution comprising:

-   -   (a) phenol in an amount of more than 50% by volume based on the        total amount of the solution;    -   (b) a polyol in an amount of 3 to 10% by volume based on the        total amount of the solution;    -   (c) a guanidinium salt at a concentration of 0.5 to 2.0 M based        on the total amount of the solution;    -   (d) a thiocyanate at a concentration of 0.1 to 0.5 M based on        the total amount of the solution;    -   (e) a buffer for maintaining the pH of the solution at 4 to 6;        and    -   (f) an organic solvent for separation of an aqueous layer;

centrifuging the obtained homogenate; and

recovering an RNA-containing aqueous layer produced by thecentrifugation.

-   [9] The method according to claim 7 or 8, wherein the phenol    concentration is 55 to 65% by volume based on the total amount of    the solution of (a) to (e).

By using the solution of the present invention, substantially pure RNAfree from contamination with DNA can be simply extracted from abiological sample. Further, by the present invention, RNA can beobtained without an additional treatment such as DNase treatment whichmay cause recovery loss, which RNA has purity that allows use of the RNAas it is even in uses wherein quantitativeness is required. Inparticular, an RNA of interest can be extracted with high purity evenfrom, among biological samples, body fluids such as blood containingvery large amounts of RNase and other contaminants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows electropherograms of nucleic acid extracted from serum inExample 1 using a solution according to an embodiment of the presentinvention.

FIG. 2 shows electropherograms of nucleic acid extracted from serum inComparative Example 1 using a solution described in Patent Document 2.

FIG. 3 shows an electropherogram of nucleic acid extracted from serum inComparative Example 2 using a solution described in Patent Document 1.

FIG. 4 shows electropherograms of nucleic acid extracted from serum inExamples 2 to 5 using solutions according to embodiments of the presentinvention.

FIG. 5 shows electropherograms of nucleic acid extracted from serum inExamples 6 to 12 using solutions according to embodiments of the presentinvention.

FIG. 6 shows electropherograms of nucleic acid extracted from serum inExample 13 and Comparative Example 3 using a solution according to anembodiment of the present invention and a solution described in PatentDocument 3.

FIG. 7 shows electropherograms of nucleic acid extracted from culturedcells in Example 14 using a solution according to an embodiment of thepresent invention.

FIG. 8 shows electropherograms of nucleic acid extracted from serum inExamples 15 and 16 using solutions according to embodiments of thepresent invention.

FIG. 9 shows electropherograms of nucleic acid extracted from serum inComparative Example 4 using a solution described in Patent Document 3.

FIG. 10 shows electropherograms of nucleic acid extracted from serum inExamples 17 and 18 using solutions according to embodiments of thepresent invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention provides a solution for extracting RNA from abiological sample, which solution comprises as its components thefollowing (a) to (e):

(a) phenol in an amount of more than 50% by volume based on the totalamount of the solution;

(b) a polyol in an amount of 3 to 10% by volume (not less than 3% byvolume and not more than 10% by volume) based on the total amount of thesolution;

(c) a guanidinium salt at a concentration of 0.5 to 2.0 M (not less than0.5 M and not more than 2.0 M) based on the total amount of thesolution;

(d) a thiocyanate at a concentration of 0.1 to 0.5 M (not less than 0.1M and not more than 0.5 M) based on the total amount of the solution;and

(e) a buffer for maintaining the pH of the solution at 4 to 6.

The biological sample used in the present invention comprises RNA and atleast DNA. Further, by using the solution of the present invention,substantially pure RNA can be extracted from the biological sample. Theterm “substantially pure RNA” herein means RNA from which DNA containedin the original biological sample has been separated and which issubstantially free from contamination with the DNA. Whether or not RNAis substantially pure can be judged by seeing whether or not DNA isdetected by electrophoresis, For example, since “Agilent RNA 6000 picokit” manufactured by Agilent Technologies Inc. (model number, 5067-1513)can be used for detection of nucleic acid in an amount of 50 pg/μL to5000 pg/μL (recommendation), the kit can be used for evaluation of thepresence/absence of DNA contamination. More specifically, the extractednucleic acid may be treated with RNase and subjected to electrophoresisusing “Agilent RNA 6000 pico kit”. In cases where no peak was detected,it can be said that DNA contamination was sufficiently suppressed andsubstantially pure RNA could be obtained. Further, by analyzing theamount of DNA contamination by quantitative PCR, the purity of RNA canbe evaluated. For example, in cases where a real-time PCR apparatus and“SYBR Green” (fluorescent dye) are used, double-stranded DNA in anamount of 60 pg can be detected, so that the evaluation can be carriedout using these. More specifically, extracted nucleic acid is added to aPCR reaction solution containing primers, DNA polymerase and “SYBRGreen” to perform PCR amplification, and the result is compared with apreliminarily prepared calibration curve. By this, the amount of DNAcontamination can be quantitatively analyzed.

In the present invention, the total amount of a solution means the totalvolume comprising all of the above-described (a) to (e). For example,“phenol in an amount of more than 50% by volume based on the totalamount of the solution” means that more than 500 mL of phenol iscontained in 1 L of the solution after mixing of all components.Further, for example, “a guanidinium salt at a concentration of 0.5 to2.0 M based on the total amount of the solution” means that the finalconcentration in the solution is not less than 0.5 M and not more than2.0 M, that is, the guanidinium salt is contained in an amount of notless than 0.5 mol and not more than 2 mol in 1 L of the solution aftermixing of all components.

The solution according to an embodiment of the present inventioncomprises (a) phenol in an amount of more than 50% by volume based onthe total amount of the solution. It was found that employing a phenolconcentration of more than 50% by volume, which is different from theconcentration employed in conventional techniques, produces the effectof reducing contamination of the aqueous layer, into which RNA isextracted, with DNA as an impurity. For example, the solution accordingto an embodiment of the present invention comprises phenol in an amountof not less than 51% by volume, not less than 52% by volume, not lessthan 53% by volume, not less than 54% by volume or not less than 55% byvolume. The solution of the present invention comprises phenol in anamount of preferably not less than 53% by volume, more preferably notless than 55% by volume. Further, the concentration of phenol ispreferably not more than 75% by volume in view of preparing the solutionof the present invention in the state where other components of thesolution of the present invention, (b) polyalcohol, (c) 0.5 to 2.0 Mguanidinium salt and (d) 0.1 to 0.5 M thiocyanate are uniformly mixed atthe respective predetermined concentrations. Further, the concentrationof phenol is more preferably not more than 65% by volume in view ofreducing the influence of oxidation of phenol. The range of the phenolconcentration is preferably one determined by an arbitrary combinationof these upper limits and lower limits, and is more preferably not lessthan 52% by volume and not more than 65% by volume, not less than 53% byvolume and not more than 65% by volume, especially preferably not lessthan 55% by volume and not more than 65% by volume.

The solution according to an embodiment of the present inventioncomprises (b) a polyol in an amount of 3 to 10% by volume based on thetotal amount of the solution. The polyol in the present invention may bean aliphatic alcohol having a plurality of hydroxyl groups, which allowsmixing of the (a) phenol component and the aqueous solutions of (c) and(d) in the solution of the present invention, to keep the solution ofthe present invention uniform. As the polyol, a C₂-C₆ aliphatic alcoholhaving 2 to 4 hydroxyl groups is preferred. Examples of the polyolinclude glycerol, ethylene glycol, propylene glycol and erythritol, andthe polyol is more preferably glycerol. The polyol may be used in anamount of 3 to 10% by volume based on the total amount of the solutionof the present invention in order to maintain the solution of thepresent invention as a uniform solution and to prevent excessivedistribution of the phenol component into the aqueous layer.

The solution according to an embodiment of the present inventioncomprises (c) a guanidinium salt at a concentration of 0.5 to 2.0 Mbased on the total amount of the solution. Specific preferred examplesof the guanidinium salt include guanidinium thiocyanate and guanidiniumhydrochloride. Guanidinium salts have an effect to protect RNA fromdegradation and to keep phenol in the solution state in an aqueoussolution.

The solution according to an embodiment of the present inventioncomprises (d) a thiocyanate at a concentration of 0.1 to 0.5 M based onthe total amount of the solution. As the thiocyanate, an inorganic saltof thiocyanic acid may be preferably used, and ammonium thiocyanate andsodium thiocyanate may be more preferably used. Further, the thiocyanatemay be a mixture of a plurality of different inorganic salts ofthiocyanic acid, and, for example, a mixture of ammonium thiocyanate andsodium thiocyanate may be preferably used. Thiocyanate is considered toenhance RNA extraction from a biological sample. In cases where thesolution of the present invention comprises guanidinium thiocyanate, theconcentration of guanidinium thiocyanate is included in theconcentration of the above-described guanidinium salt, and not includedin the concentration of thiocyanate.

The solution according to an embodiment of the present inventioncomprises (e) a buffer for maintaining the pH of the solution at 4 to 6.As the buffer, organic salts and inorganic salts which areconventionally used for maintaining the pH within a desired range andshow buffering capacity may be used. Specific examples of the bufferinclude organic salts and inorganic salts, such as phosphate, acetate,citrate, phthalate, tartrate and lactate, of sodium, potassium, lithiumand ammonium. Among the combinations of these, sodium acetate and sodiumcitrate are more preferably used. Further, a plurality of these organicsalts and/or inorganic salts may be used in combination. Theconcentration of the buffer is not restricted as long as it issufficient for maintaining the pH within the desired range of 4 to 6,and the concentration is preferably 0.02 to 0.2 M based on the totalamount of the solution of the present invention. In order to adjust thepH of the solution of the present invention, an appropriate aqueous acidor alkaline solution such as a hydrochloric acid or sodium hydroxidesolution may be added as appropriate in addition to the buffer.

The solution of the present invention may contain a surfactant(s) suchas polyoxyethylene sorbitan, sodium dodecyl sulfate and/or sarcosine forsupporting purification of the RNA of interest by denaturing proteins inthe biological sample. Further, the solution of the present inventionmay contain an antioxidant(s) such as hindered amine phenol and/orquinoline for prevention of oxidation of phenol.

In cases where the biological sample is in the liquid state when the RNAof interest is to be extracted, the solution of the present inventionmay be used in an amount of not less than 1 volume, preferably not lessthan 3 volumes of the sample.

An example of the procedure for extraction of the RNA of interest usingthe solution of the present invention is shown below. First, thebiological sample is homogenized in the solution of the presentinvention to form a homogenate. The method of homogenization is notrestricted, and examples of the method include stirring by vortexing orthe like, crushing with an injection needle or the like, and use of aconventional homogenizer. Subsequently, an organic solvent is added tothe homogenate for separation of the aqueous layer, and the resultingmixture is subjected to centrifugation. The organic solvent to be addedin this step is preferably used in an amount of about 2% by volume toabout 40% by volume based on the homogenate. The centrifugation may becarried out usually at 6,000×G to 20,000×G for 3 minutes to 30 minutes,for example, at a rate of 12,000×G for 10 minutes at room temperature.However, the rate, temperature and time are not restricted as long asthe aqueous layer can be separated. By the centrifugation, thesubstantially pure RNA of interest is extracted into the aqueous layer.On the other hand, DNA, proteins and the like are separated into theorganic layer, or, in cases where an intermediate layer was produced,DNA, proteins and the like are separated into the organic layer and theintermediate layer.

The organic solvent for separation of the aqueous layer is a liquidorganic compound to be used for achieving separation into the aqueouslayer comprising the RNA of interest extracted using the solution of thepresent invention and the organic layer and/or the intermediate layer(if produced) comprising DNA and the like. As this organic solvent, onewhich has the same degree of hydrophilicity as, or is more hydrophobicthan, phenol may be used. For example, in terms of the water/octanoldistribution coefficient CLogP which is commonly used as an index ofhydrophilicity, an organic compound having a value of not less than 1.4(CLogP value for phenol) may be used, and an organic compound having avalue within the range of 1.4 to 5 may be preferably used. An estimatedvalue of the CLogP value can be calculated by, for example, using aprogram such as “Chem Draw” (registered trademark). Examples of theorganic solvent which may be used in the present invention include, butare not limited to, chloroform (1.952), p-bromoanisole (3.064),1-bromo-3-chloropropane (1.847), 4-bromoveratrole (2.7345),6-bromo-1,4-benzodioxane (3.0005), 1-bromo-4-trifluoromethoxybenzene(4.173), 1-bromo-2,4-dimethoxybenzene (2.8545), 4-fluoroanisole (2.344),4-bromotoluene (3.504) and ethyl 4-bromobutyrate (1.772). The value inthe parentheses for each of the above organic solvents indicates theCLogP value calculated with “Chem Draw”.

The organic solvent for separation of the aqueous layer may be used byformation using the solution of the present invention comprising (a) to(e) as described above and addition to the homogenate, but the organicsolvent may also be preliminarily contained in the solution of thepresent invention comprising the above-described (a) to (e). In cases ofa conventional solution whose phenol concentration is not more than 50%,inclusion of this organic solvent in advance causes separation of thesolution into the aqueous layer and the organic layer before mixing witha biological sample, so that it has been difficult to use the solutionas an extraction solution. In contrast, at the phenol concentration ofthe solution of the present invention, the organic solvent can beuniformly mixed with the solution of the present invention, and theresulting solution can be stored as a single solution. In cases of thesolution of the present invention preliminarily containing the organicsolvent, separation of the aqueous phase containing RNA is possible byadding a biological sample to the solution and homogenizing theresulting mixture to provide a homogenate, followed by immediatelysubjecting the homogenate to centrifugation. Accordingly, compared tothe cases where addition of the organic solvent to the homogenate iscarried out later, the procedure can be made very simple, which ispreferred.

In cases where the organic solvent for separation of the aqueous layeris preliminarily contained in the solution of the present inventioncomprising the above-described (a) to (e), the content of the organicsolvent may be selected depending on the type of the organic solvent tobe added and the phenol concentration in the solution, within the rangein which the organic solvent can be uniformly mixed in the solution ofthe present invention. For example, in cases where the phenolconcentration in the solution of the present invention is 65% andchloroform is selected as the organic solvent, chloroform is containedpreferably in an arbitrary volume of up to 27% by volume based on thetotal amount, 100%, of the solution comprising the above-described (a)to (e). More specifically, it is preferred to add chloroform in anarbitrary volume of up to 27 mL, to 100 mL of the solution comprisingthe above-described (a) to (e). Chloroform is contained in an amount ofmore preferably 5 to 25% by volume, still more preferably 10 to 20% byvolume based on the total amount, 100%, of the solution comprising theabove-described (a) to (e). Further, in cases where the phenolconcentration is 58%, chloroform is contained preferably in an arbitraryvolume of up to 14%, more preferably in an amount of 6 to 13% by volume,still more preferably in an amount of 8 to 12% by volume based on thetotal amount, 100%, of the solution comprising the above-described (a)to (e). Further, in cases where the phenol concentration in the solutionis 65% and p-bromoanisole is selected as the organic solvent,p-bromoanisole is contained preferably in an arbitrary volume of up to22% by volume, more preferably in an amount of 5 to 20% by volume, stillmore preferably in an amount of 10 to 18% by volume based on the totalamount, 100%, of the solution comprising the above-described (a) to (e).Further, similarly, in cases where the phenol concentration is 58%,p-bromoanisole is contained preferably in an arbitrary volume of up to13% by volume, more preferably in an amount of 3 to 11% by volume, stillmore preferably in an amount of 5 to 9% by volume based on the totalamount, 100%, of the solution comprising the above-described (a) to (e).

For further purifying and concentrating the RNA extracted into theaqueous layer using the solution of the present invention, a loweralcohol may be added to the aqueous layer comprising RNA in order toprecipitate the RNA, and the precipitated RNA may be recovered.Alternatively, the RNA precipitated by addition of a lower alcohol tothe aqueous layer comprising RNA may be adsorbed to a carrier to whichRNA can be adsorbed, such as a silica membrane column, and the RNA maythen be eluted and recovered from the carrier (column). Examples of thelower alcohol to be used in this step include ethanol and isopropanol.The concentration of the lower alcohol may be determined according tothose employed in conventional techniques such as ethanol precipitationand isopropanol precipitation, or according to the concentrationsrecommended by manufacturers of carriers such as silica membranecolumns.

The solution of the present invention can be produced by mixing theabove-described (a) to (e) such that their respective concentrations areattained. The procedure of the mixing is not restricted. Depending onthe composition of the solution, the respective solutions at higherconcentrations may be prepared in advance before mixing the solutions.For example, 6 M aqueous guanidinium thiocyanate solution, 6 M aqueousammonium thiocyanate solution and 1 M sodium acetate may be prepared inadvance and then mixed to attain the concentrations of interest,followed by addition of glycerol, phenol and a necessary amount of waterthereto, to prepare the solution of the present invention. The solutionof the present invention wherein an organic solvent for separation ofthe aqueous layer is preliminarily contained in the solution comprisingthe above-described (a) to (e) can also be similarly produced by mixing(a) to (e) and the organic solvent such that their desiredconcentrations are attained.

The biological sample to be used in the present invention is notrestricted as long as it comprises RNA and at least DNA. For example,the biological sample may comprise, in addition to DNA, proteins asimpurity components which are preferably separated from the RNA ofinterest. Specific examples of the biological sample include culturedcells; culture liquids of cultured cells; body tissues such as surgicalsections and biopsy samples; living cells; blood; blood components(serum, plasma); urine; and body fluids such as saliva and tears. Thebiological sample is not restricted to these, and an arbitrary samplecontaining RNA may be used. When the solution of the present inventionis applied to these biological samples, in cases where the biologicalsample is a liquid sample such as a body fluid, the collected sample maybe mixed as it is with the solution of the present invention or may bediluted with PBS or water before mixing with the solution of the presentinvention. In cases where the biological sample is a cell pellet or atissue piece, the collected sample may be mixed as it is with thesolution of the present invention or may be diluted with PBS or waterbefore mixing with the solution of the present invention, and, in caseswhere the sample is diluted, a homogenate of the biological sample ispreferably prepared before dilution with water or PBS in order toprevent degradation of RNA.

Among the biological samples, body fluids, especially blood, sometimescontain a very large amount of RNase and other contaminants, and, insuch a case, extraction of substantially pure RNA by a conventionalmethod is very difficult. With a phenol concentration of more than 50%by volume, the solution of the present invention enables effectiveextraction of contaminants such as proteins into an organic layer, sothat the RNA of interest can be obtained with high purity. Further, theintermediate layer that appears after centrifugation is reduced andclear separation into layers can be achieved, so that the aqueous layercomprising the RNA of interest can be easily separated.

The RNA extracted using the solution is ribonucleic acid wherein aplurality of ribonucleotides are linked by phosphodiester bonds, and themolecular weight, the number of bases and the origin of the RNA are notrestricted. In general, RNA is classified into many types according tofunctional classification, and examples of the types include mRNA(messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), ncRNA(non-coding RNA), snRNA (small nuclear RNA) and snoRNA (small nucleolarRNA). However, in view of the chemical structure, the only knowndifference is the molecular weight (number of bases), and RNA having anymolecular weight is included in the present invention. RNAs having basenumbers of about 15 to 500 bases, which are generally called small RNAs,and RNAs generally having base numbers of about 18 to 25, which aremiRNAs (microRNAs), are also included in the RNA of the presentinvention.

In general, the main difference in the primary chemical structurebetween RNA and DNA is the presence/absence of the hydroxyl group (—OH)at the 2′-position of ribose as the constituent sugar. The smaller thenumber of bases, the smaller the structural difference between RNA andDNA and the more difficult separation of RNA and DNA by extraction.However, by use of the solution of the present invention, RNA having arelatively small number of bases such as small RNA can also be extractedwith high purity.

In the state where DNA and RNA coexist, it is usually difficult todistinguish between these and to quantify each of these using anabsorptiometer or luminometer. However, by using the solution of thepresent invention, substantially pure RNA can be obtained, so thatquantification of RNA using an absorptiometer or luminometer ispossible. Further, in cases where the solution of the present inventionis used in RNA analysis using qRT-PCR or a microarray, the analysis canbe simply carried out without requirement of treatment with DNase, inthe absence of the noise due to coexistence of DNA.

EXAMPLES

The present invention will now be described concretely by way ofExamples below. However, the scope of the present invention is notrestricted by these Examples.

Example 1 (1) Preparation of Solution for RNA Extraction

The respective components of the solution were mixed such that theirfinal concentrations were as described below, to prepare a solution forRNA extraction.

-   -   58 vol % Phenol    -   5 vol % Glycerol    -   0.8 M Guanidinium thiocyanate (mixed as an aqueous solution)    -   0.4 M Ammonium thiocyanate (mixed as an aqueous solution)    -   0.1 M Sodium acetate buffer (mixed as an aqueous solution),        adjusted to pH 5.        (2) RNA Extraction from Biological Sample

As a biological sample containing RNA as well as DNA and proteins, serumwas used for RNA extraction. By mixing 900 μL of the solution preparedin the above (1) and 300 μL of serum by vortexing, the sample washomogenized. To the resulting homogenate, 60 μL of p-bromoanisole wasadded, and the resulting mixture was mixed, followed by centrifuging themixture at room temperature at 12,000×G for 10 minutes. By this, anaqueous layer containing RNA, and an organic layer and an intermediatelayer containing DNA and proteins were formed. From these, 4004 of theaqueous layer was separated into another tube.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

To the aqueous layer containing RNA separated in (2), 1.5 volumes of100% ethanol was added, and 700 μL of the resulting mixture was placedin a column for purification of nucleic acid, “RNeasy Mini Spin Column”contained in “miRNeasy mini kit” (manufactured by QIAGEN), followed bycentrifuging the column at 8,000×G for 15 seconds to allow adsorption ofnucleic acid to the column. The liquid that passed through the columnwas discarded. By repeating this operation until no ethanol-mixed RNAsample is remaining, all nucleic acid contained in the aqueous layer wasadsorbed to the column. Thereafter, according to the protocol for“miRNeasy mini kit”, the column was washed twice with 700 μL of BufferRWT and 500 μL of Buffer RPE, and the column was then dried, followed byelution with 30 μL of RNase-free water, to obtain a purified andconcentrated RNA sample.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

In order to confirm that the extracted nucleic acid is RNA, the sampleseparated in (2) was subjected to RNase treatment. To the aqueous layercontaining RNA separated in (2), 1.5 volumes of 100% ethanol was added,and nucleic acid was allowed to adsorb to the column in the same manneras in (3a). After washing the column with 350 μL of Buffer RWT, dilutedRNase was added thereto to perform RNase treatment of the nucleic acidadsorbed to the column, and the column was washed twice with 350 μL ofBuffer RWT and 500 μL of Buffer RPE, followed by drying the column.Thereafter, elution was carried out with 30 μL of RNase-free water, toobtain a purified and concentrated RNA sample.

(4) Evaluation of Purity by Electrophoresis

After heat denaturation of 14 each of the RNA samples obtained in (3a)and (3b) at 70° C. for 2 minutes, each sample was rapidly cooled. Thesamples were then subjected to electrophoresis using “Agilent RNA 6000pico kit” manufactured by Agilent Technologies Inc. (model number,5067-1513). The results are shown in FIG. 1. Further, by the SmearAnalysis function of “Bioanalyzer 2100”, the peak area of 25 to 500 ntwas calculated to confirm the peak size and the amount (concentration)of nucleic acid detected.

In the sample (3a) wherein enzyme treatment was not carried out, only asingle peak having a size of less than 200 bases was found (lane 1). Theamount of nucleic acid calculated in this case was 816 pg/μL. On theother hand, in the electrophoretic pattern of the RNase-treated sample(3b), no peak was detected (lane 2). The amount of nucleic acidcalculated in this case was 61 pg/μL. In order to confirm the noise inthe detection system, the same operation as in Example 1 was carried outusing PBS containing no nucleic acid, instead of serum (lane 3, BLANK).Since the amount of nucleic acid calculated in this case was 63 pg/μL,the amount of nucleic acid calculated for lane 2 was considered to bedue to the noise. From the above results, the extracted nucleic acidcould be confirmed to be RNA which does not contain DNA. Since RNAhaving 22 to 25 bases and the peak obtained in the present Exampleshowed similar migration distances in electrophoresis, the RNA found inlane 1 was considered to have 22 to 25 bases.

The above results are summarized in Table 1.

Comparative Example 1 (1) Preparation of Solution for RNA Extraction

The solution described in Patent Document 2 was prepared with the samecomposition as in Example 1 except that the phenol concentration was 50%by volume in terms of the final concentration of the solution.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(3c) Purification and Concentration of RNA from Aqueous Layer—With DNaseTreatment—

The operation was carried out in the same manner as in (3b) in Example 1except that the aqueous layer containing RNA was treated with DNaseinstead of the RNase in (3b), to obtain a purified and concentratedsample. Other conditions were the same as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in FIG. 2.

In the sample wherein enzyme treatment was not carried out, two strongpeaks (corresponding to numbers of bases of about 200 and about 500) andone weak peak (corresponding to the same number of bases as inExample 1) were detected (lane 1). In the sample treated with RNase, thepeaks of 200 bases and 500 bases hardly changed, and it was thereforefound that the two peaks were not due to RNA (lane 2). On the otherhand, the single weak peak has disappeared, and this peak was thereforeconfirmed to be due to RNA as in the case of Example 1. In theDNase-treated sample, the two strong peaks have disappeared, and veryshort fragments due to degradation were detected (lane 3). Therefore,these two strong peaks were found to be due to contamination with DNAfragments.

Thus, when the solution containing 50% by volume of phenol was used,contamination with DNA was observed, and pure RNA could not beextracted.

The above results are summarized in Table 3.

Comparative Example 2 (1) Preparation of Solution for RNA Extraction

The same solution as the extraction solution described in PatentDocument 1 was prepared except that the phenol concentration was 60% byvolume. That is, the solution contained 60% by volume of phenol, 2Mguanidinium thiocyanate, 0.1 M sodium acetate and 0.2% by volume of2-mercaptoethanol in terms of the final concentrations, and the pH ofthe solution was 4.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in FIG. 3.

Three peaks similar to those in Comparative Example 1 were observed.Therefore, contamination with DNA fragments could be confirmed. Theabove result is summarized in Table 3.

Example 2 (1) Preparation of Solution for RNA Extraction

A solution was prepared such that the composition of the solution is thesame as in Example 1 except that the phenol concentration was 55% byvolume in terms of the final concentration.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in FIG. 4.

The same peak as in Example 1 was detected (lane 1), and it could beconfirmed that only RNA was extracted with high purity. Since the peakin the RNase-treated sample (lane 5; RNase (+)) was as weak as that inBLANK, it could be confirmed that the extracted nucleic acid containedonly RNA.

The above results are summarized in Table 1.

Example 3 (1) Preparation of Solution for RNA Extraction

A solution was prepared such that the composition of the solution is thesame as in Example 1 except that the phenol concentration was 65% byvolume in terms of the final concentration.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 2 in FIG. 4.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 4 (1) Preparation of Solution for RNA Extraction

A solution was prepared such that the composition of the solution is thesame as in Example 1 except that the phenol concentration was 53% byvolume in terms of the final concentration.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 3 in FIG. 4.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 5 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 240 μL of chloroform wasadded instead of 60 μL of p-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 4 in FIG. 4.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 6 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of 4-bromoveratrolewas added instead of 60 μL of p-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 1 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 7 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of6-bromo-1,4-benzodioxane was added instead of 60 μL of p-bromoanisole tothe homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 2 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 8 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of1-bromo-4-trifiuoromethoxybenzene was added instead of 60 μL ofp-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 3 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 9 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of1-bromo-2,4-dimethoxybenzene was added instead of 60 μL ofp-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 4 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 1.

Example 10 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of 4-fluoroanisolewas added instead of 60 μL of p-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 5 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 2.

Example 11 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of 4-bromotoluene wasadded instead of 60 μL of p-bromoanisole to the homogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 6 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 2.

Example 12 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample, except that 100 μL of ethyl4-bromobutyrate was added instead of 60 μL of p-bromoanisole to thehomogenate.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 7 in FIG. 5.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity.

The above result is summarized in Table 2.

Example 13 (1) Preparation of Solution for RNA Extraction

A solution was prepared by adding hydrochloric acid to the solutionprepared in Example 1 such that the pH of the solution was adjusted to4.2.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 1 in FIG. 6.

The same peak as in Example 1 was detected also in the case where the pHof the solution was 4.2, and it could be confirmed that only RNA wasextracted with high purity.

The above result is summarized in Table 2.

Comparative Example 3 (1) Preparation of Solution for RNA Extraction

A solution was prepared by adding hydrochloric acid to the solutionprepared in Comparative Example 1 such that the pH of the solution wasadjusted to 3.6.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresult is shown in lane 2 in FIG. 6. The same three peaks as inComparative Example 1 were observed. By this, it could be confirmedthat, in cases where the solution containing 50% by volume of phenol isused, contamination with DNA fragments occurs also at a pH of 3.6.

The above result is summarized in Table 3.

Example 14 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 was prepared.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 exceptthat cultured cells (HEK293 cells) suspended in 300 μL of PBS were usedas the biological sample, instead of 300 μL of serum.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1 exceptthat 1.25 volumes, instead of 1.5 volumes, of ethanol was added to theaqueous layer.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1 exceptthat 1.25 volumes, instead of 1.5 volumes, of ethanol was added to theaqueous layer.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1 exceptthat “Agilent RNA 6000 nano kit” (model number, 5067-1511) (manufacturedby Agilent Technologies Inc.) was used as the kit, instead of “AgilentRNA 6000 pico kit”. The results are shown in FIG. 7.

It could be confirmed that, in the sample without enzyme treatment in(3a), 185 and 28S ribosome RNAs were extracted almost withoutdegradation (RIN value: 2.3) (lane 1). The amount of nucleic acidcalculated in this case was 79 ng/μL. In the result obtained with theRNase-treated sample in (3b), the same peak as in Example 1 was notdetected at all (lane 2). The amount of nucleic acid calculated in thiscase was 4 ng/μL. The noise produced in the detection system when nosample was subjected to electrophoresis was investigated (lane 3) and,as a result, the amount of nucleic acid in this case was 2 ng/μL. Thus,the amount of nucleic acid in lane 2 was considered to be due to thenoise. Based on these results, the extracted nucleic acid was confirmedto be entirely RNA.

The above results are summarized in Table 2.

Examples 15 (1) Preparation of Solution for RNA Extraction

The respective components of the solution were mixed such that theirfinal concentrations were as described below, to prepare a solution forRNA extraction. That is, 60 μL of additional p-bromoanisole was added to900 μL of the solution having the same composition as in Example 1, toprepare the solution.

-   -   58 vol % Phenol    -   5 vol % Glycerol    -   0.8 M Guanidinium thiocyanate (mixed as an aqueous solution)    -   0.4 M Ammonium thiocyanate (mixed as an aqueous solution)    -   0.1 M Sodium acetate buffer (mixed as an aqueous solution),        adjusted to pH 5.    -   6.6 vol % p-Bromoanisole based on the total amount (100%) of the        above components        (2) RNA Extraction from Biological Sample

By mixing 900 μL of the solution prepared in the above (1) and 300 μL ofserum by vortexing, the sample was homogenized. The resulting homogenatewas centrifuged at room temperature at 12,000×G for 10 minutes. By this,an aqueous layer containing RNA, and an organic layer and anintermediate layer containing DNA and proteins were formed. From these,350 μL of the aqueous layer was separated into another tube.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in lanes 1, 3 and 5 in FIG. 8.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity (lane 1). Since the peak inthe RNase-treated sample (lane 3; RNase (+)) was as weak as that inBLANK (lane 5), it could be confirmed that the extracted nucleic acidcontained only RNA.

The above results are summarized in Table 2.

Examples 16 (1) Preparation of Solution for RNA Extraction

The respective components of the solution were mixed such that theirfinal concentrations were as described below, to prepare a solution forRNA extraction. That is, 90 μL of additional chloroform was added to 900μL of the solution having the same composition as in Example 1, toprepare the solution.

-   -   58 vol % Phenol    -   5 vol % Glycerol    -   0.8 M Guanidinium thiocyanate (mixed as an aqueous solution)    -   0.4 M Ammonium thiocyanate (mixed as an aqueous solution)    -   0.1 M Sodium acetate buffer (mixed as an aqueous solution),        adjusted to pH    -   10 vol % chloroform based on the total amount (100%) of the        above components        (2) RNA Extraction from Biological Sample

By mixing 900 μL of the solution prepared in the above (1) and 300 μL ofserum by vortexing, the sample was homogenized. The resulting homogenatewas centrifuged at room temperature at 12,000×G for 10 minutes. By this,an aqueous layer containing RNA, and an organic layer and anintermediate layer containing DNA and proteins were formed. From these,350 μL of the aqueous layer was separated into another tube.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in lanes 2, 4 and 5 in FIG. 8.

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity (lane 2). Since the peak inthe RNase-treated sample (lane 4; RNase (+)) was as weak as that inBLANK (lane 5), it could be confirmed that the extracted nucleic acidcontained only RNA.

The above results are summarized in Table 2.

Comparative Example 4 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Comparative Example 3 wasprepared except that the phenol concentration was 55% by volume.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in FIG. 9.

In the sample without enzyme treatment in (3a), a rather broader peakcompared to the peak in Example 1 was detected (lane 1), and theRNase-treated sample in (3b) also showed a peak (lane 2). Since the peakobtained with the RNase-treated sample (RNase(+)) was also broader thanthe peak in BLANK (lane 2), contamination with nucleic acid other thanRNA (DNA) could be confirmed.

The above results are summarized in Table 3.

Example 17 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 in terms of thefinal concentrations was prepared, except that the pH of was adjusted to4.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in FIG. 10 (lanes 1, 2 and 5).

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity (lane 1). Since the peak inthe RNase-treated sample (lane 2; RNase (+)) was as weak as that inBLANK (lane 5), it could be confirmed that the extracted nucleic acidcontained only RNA.

The above results are summarized in Table 2.

Example 18 (1) Preparation of Solution for RNA Extraction

The solution having the same composition as in Example 1 in terms of thefinal concentrations was prepared, except that the pH was adjusted to 6.

(2) RNA Extraction from Biological Sample

The operation was carried out in the same manner as in Example 1 usingserum as the biological sample.

(3a) Purification and Concentration of RNA from Aqueous Layer—WithoutEnzyme Treatment—

The operation was carried out in the same manner as in Example 1.

(3b) Purification and Concentration of RNA from Aqueous Layer—With RNaseTreatment—

The operation was carried out in the same manner as in Example 1.

(4) Evaluation of Purity by Electrophoresis

The operation was carried out in the same manner as in Example 1. Theresults are shown in FIG. 10 (lanes 3, 4 and 5).

The same peak as in Example 1 was detected, and it could be confirmedthat only RNA was extracted with high purity (lane 3). Since the peak inthe RNase-treated sample (lane 4; RNase (+)) was as weak as that inBLANK (lane 5), it could be confirmed that the extracted nucleic acidcontained only RNA.

The above results are summarized in Table 2.

TABLE 1 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6Example 7 Example 8 Example 9 Composition of solution Phenol (% byvolume) 58 55 65 53 58 58 58 58 58 Guanidinium thiocyanate (M) 0.8 0.80.8 0.8 0.8 0.8 0.8 0.8 0.8 Ammonium thiocyanate (M) 0.4 0.4 0.4 0.4 0.40.4 0.4 0.4 0.4 Glycerol (% by volume) 5 5 5 5 5 5 5 5 5 Sodium acetate(M) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 2-Mercaptoethanol (% by — — — —— — — — — volume) pH of solution 5 5 5 5 5 5 5 5 5 Organic solvent(added later) Bromoan- Bromoan- Bromoan- Bromoan- Chloroform Bromovera-Benzo- Trifluoro- Dimethoxy isole isole isole isole trole dioxanemethoxy- benzene benzene Organic solvent (preliminarily — — — — — — — —— contained) Biological sample Serum Serum Serum Serum Serum Serum SerumSerum Serum Amount of nucleic acid extracted (pg/μL) Without enzymetreatment 816 421 With RNase treatment 61 37 With DNase treatment — —Blank 63 41 Electropherogram FIG. 1 FIG. 4 FIG. 4 FIG. 4 FIG. 4 FIG. 5FIG. 5 FIG. 5 FIG. 5 Figure number/lane number Lane 1, 2 Lane 1, 5 Lane2 Lane 3 Lane 4 Lane 1 Lane 2 Lane 3 Lane 4 Purity of RNA extracted goodgood good good good good good good good

TABLE 2 Example Example Example Example Example Example Example ExampleExample 10 11 12 13 14 15 16 17 18 Composition of solution Phenol (% byvolume) 58 58 58 58 58 58 58 58 58 Guanidinium thiocyanate (M) 0.8 0.80.8 0.8 0.8 0.8 0.8 0.8 0.8 Ammonium thiocyanate (M) 0.4 0.4 0.4 0.4 0.40.4 0.4 0.4 0.4 Glycerol (% by volume) 5 5 5 5 5 5 5 5 5 Sodium acetate(M) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 2-Mercaptoethanol (% by — — — —— — — — — volume) pH of solution 5 5 5 4.2 5 5 5 4 6 Organic solvent(added later) Fluoroan- Bromo- Ethyl Bromoan- Bromoan- — — Bromoan-Bromoan- isole toluene Bromobutyrate isole isole isole isole Organicsolvent (preliminarily — — — — — Bromoan- Chloroform — — contained)isole Biological sample Serum Serum Serum Serum Cultured Serum SerumSerum Serum cells Amount of nucleic acid extracted (pg/μL) Withoutenzyme treatment 274 209 193 174 With RNase treatment 24 29 25 22 WithDNase treatment — — — — Blank 41 41 38 38 Electropherogram FIG. 5 FIG. 5FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 8 FIG. 10 FIG. 10 Figure number/lanenumber Lane 5 Lane 6 Lane 7 Lane 1 Lane 1, 2 Lane 1, 3 Lane 2, 4 Lane 1,2 Lane 3, 4 Purity of RNA extracted good good good good good good goodgood good

TABLE 3 Comparative Comparative Comparative Comparative Example 1Example 2 Example 3 Example 4 Composition of solution Phenol (% byvolume) 50 60 50 55 Guanidinium thiocyanate (M) 0.8 2 0.8 0.8 Ammoniumthiocyanate (M) 0.4 — 0.4 0.4 Glycerol (% by volume) 5 — 5 5 Sodiumacetate (M) 0.1 0.1 0.1 0.1 2-Mercaptoethanol (% by — 0.2 — — volume) pHof solution 5 4 3.6 3.6 Organic solvent (added later) BromoanisoleBromoanisole Bromoanisole Bromoanisole Organic solvent (preliminarily —— — — contained) Biological sample Serum Serum Serum Serum Amount ofnucleic acid extracted (pg/μL) Without enzyme treatment 4796 302 WithRNase treatment 784 78 With DNase treatment 499 — Blank 42 54Electropherogram FIG. 2 FIG. 3 FIG. 6 FIG. 9 Figure number/lane numberLane 1-3 Lane 1 Lane 2 Lane 1, 2 Purity of RNA extracted not good notgood not good not good

1. A solution for extracting RNA from a biological sample containing RNAand at least DNA, said solution comprising: (a) phenol in an amount ofmore than 50% by volume based on the total amount of said solution; (b)a polyol in an amount of 3 to 10% by volume based on the total amount ofsaid solution; (c) a guanidinium salt at a concentration of 0.5 to 2.0 Mbased on the total amount of said solution; (d) a thiocyanate at aconcentration of 0.1 to 0.5 M based on the total amount of saidsolution; and (e) a buffer for maintaining the pH of said solution at 4to
 6. 2. The solution according to claim 1, wherein the phenolconcentration is 55 to 65% by volume based on the total amount of saidsolution.
 3. The solution according to claim 1, further comprising anorganic solvent for separating an aqueous layer.
 4. The solutionaccording to claim 1, wherein said biological sample is a culture liquidof cultured cells.
 5. The solution according to claim 1, wherein saidbiological sample is a body fluid component of an organism.
 6. Thesolution according to claim 1, wherein said biological sample is a bloodcomponent of an organism.
 7. A method for extracting RNA from abiological sample containing RNA and at least DNA, said methodcomprising the steps of: homogenizing said biological sample togetherwith a solution comprising: (a) phenol in an amount of more than 50% byvolume based on the total amount of said solution; (b) a polyol in anamount of 3 to 10% by volume based on the total amount of said solution;(c) a guanidinium salt at a concentration of 0.5 to 2.0 M based on thetotal amount of said solution; (d) a thiocyanate at a concentration of0.1 to 0.5 M based on the total amount of said solution; and (e) abuffer for maintaining the pH of said solution at 4 to 6; mixing theobtained homogenate with an organic solvent for separation of an aqueouslayer; centrifuging the obtained mixture; and recovering anRNA-containing aqueous layer produced by the centrifugation.
 8. A methodfor extracting RNA from a biological sample containing RNA and at leastDNA, said method comprising the steps of: homogenizing said biologicalsample together with a solution comprising: (a) phenol in an amount ofmore than 50% by volume based on the total amount of said solution; (b)a polyol in an amount of 3 to 10% by volume based on the total amount ofsaid solution; (c) a guanidinium salt at a concentration of 0.5 to 2.0 Mbased on the total amount of said solution; (d) a thiocyanate at aconcentration of 0.1 to 0.5 M based on the total amount of saidsolution; (e) a buffer for maintaining the pH of said solution at 4 to6; and (f) an organic solvent for separation of an aqueous layer;centrifuging the obtained homogenate; and recovering an RNA-containingaqueous layer produced by the centrifugation.
 9. The method according toclaim 7 or 8, wherein the phenol concentration is 55 to 65% by volumebased on the total amount of said solution of (a) to (e).